The proposed aims build on investigations during the previous funding period on the identification of a single ligand-receptor pair, EphB1-ephrinB2 that directs repulsion of ventrotemporal (VT) retinal ganglion cell (RGC) growth cones at the optic chiasm midline. EphB1, expressed strictly on VT RGCs, interacts with the ligand ephrinB2 on radial glia at the chiasm midline to direct formation of the uncrossed projection. Mechanisms of midline crossing that guide RGC axons across the optic chiasm midline remain unclear. We have identified NrCAM as necessary for the establishment of the late crossed projection from VT retina. NrCAM is also expressed in RGCs outside the VT crescent. In Aim 1.a, the function of two other CAMs, TAG- 1 and Neurofascin, expressed similarly to NrCAM, will be investigated for their involvement in crossing the midline. In addition, we localized Sema6D at the chiasm midline and its receptor, Plexin-A1, is expressed in all crossing RGCs. In vitro, Sema6D inhibits the growth of RGCs that project across the midline. In Aim 1.b., the actions of Sema6D and Plexin-A1 in mediating the crossed RGC projection will be studied. CAMs have been shown to collaborate with semaphorins in other crossing systems. This issue will be addressed in Aim 1.c. Transgenic mice, culture assays, and methods for gene delivery (electroporation in utero and ex utero) developed in our lab will be used to provide a comprehensive view of the crossing process in the chiasm. The formation of eye-specific terminations in the dLGN is a direct consequence of retinal axon decussation in the optic chiasm. In our studies on the uncrossed projection, we asked how aberrant midline decussation affects specific innervation patterns in the dLGN. In EphB1 gain- and loss-of-function models, RGCs misproject to the wrong side of the midline yet terminate in appropriate eye-specific zones. These results implicate molecular tags for eye-specific innervation. As a foundation for further analysis, in Aim 2.a., we will chronicle the ingrowth, arbor formation and segregation of mouse retinogeniculate axons at the single-fiber level, as this information is lacking. In Aim 2.b., we will identify molecules that may implement eye-specific innervation of the dLGN, by 1) localization of candidates studied in Aim 1, and 2) transcriptional profiling of specific tissue regions. In Aim 2.c. and d., we will then determine the role of candidate molecules by perturbing them and by altering neural activity. The phenomenon of coordinate action by multiple guidance families such as Semaphorins and CAMs, presents a new level of analysis of axon navigation in higher vertebrate retinal pathways, where divergence is controlled by two distinct molecular systems. The proposed experiments thus address fundamental questions on how the binocular pathways develop.